The histamine H₄ receptor (H₄R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H₄R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H₄R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H₄R in comparison to human H₄R expressed in Sf9 insect cells. Only hH₄R and cH₄R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH₄R exhibited lower ligand-affinities than hH₄R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH₄R than at other H₄R species orthologs. Unlike the other H₄R species orthologs, hH₄R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH₄R, rH₄R, and mH₄R, whereas at hH₄R, JNJ7777120 was a partial inverse agonist. H₄R agonists from the class of N ( G )-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H₄R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H₄R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H₄R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of "the prototypical H₄R antagonist" JNJ7777120 as H₄R antagonism.